Project Summary/Abstract Overactivation of the Ras oncogene is a common trait of more than one third of all tumors and promotes oncogenic transformation, cell survival, growth and proliferation, and cell cycle progression. Therefore, significant efforts have been focused on developing therapeutics that target Ras and its downstream effectors; however, the therapeutics are not effective in all tumors and cells, have severe side effects, and the tumors often adapt and develop resistance. However, multiple studies have revealed that cancer cells, including Ras- driven tumors, acquire unique vulnerabilities as a consequence of adapting cellular mechanisms that promote uncontrolled proliferation and suppress apoptosis. Targeting these vulnerabilities provides an opportunity to develop novel therapeutics. Previously, we have discovered that Kinase Suppressor of Ras 1 (KSR1) acts as a scaffold and modulates downstream Ras effectors. Our lab has demonstrated that KSR1 is required for Ras- driven transformation, and depletion of Kinase Suppressor of Ras 1 (KSR1) by RNA interference (RNAi) selectively kills malignant, Ras-driven cancer cells, but does not kill immortalized, non-transformed human colon epithelial cells (HCECs). Finally, KSR1-/- mice are fertile and phenotypically normal (with the only exception being a minor hair follicle defect) suggesting KSR1 is not required for normal cell survival. This evidence suggests that KSR1 represents a Ras-driven vulnerability that could be targeted for therapy without significant side effects. Therefore, KSR1, its downstream effectors, and functional analogues of KSR1 are potential targets for therapy in Ras-driven tumors and are likely to offer a high therapeutic index. To identify other genes that are similar to KSR1 (i.e., are required for the survival of Ras-driven cancer cells, but not normal cells), we performed a gene expression-based high-throughput screen. By analyzing the genome-scale screen data, I hypothesize that we will identify novel vulnerabilities in Ras-driven cancers and associated potential therapeutic targets by characterizing the identified KSR1-like hits. To evaluate this hypothesis, potential therapeutic targets in Ras-mutated tumors were identified by analyzing our functional genome-scale RNAi screen. The first two targets examined have been biologically validated with additional targets undergoing biological validation to determine the extent to which they are required for Ras- driven cancer cell survival in vitro (Aim #1). TIMELESS, a biologically validated hit, will be characterized to evaluate its selective requirement in cancer and assess its potential to serve as a target for Ras-mutated cancer therapeutics in vivo (Aim #2). This project will provide a platform for identifying novel therapeutic targets, highlight the benefit of applying bioinformatics to enrich the results, and establish the ability to identify oncogene-specific vulnerabilities from an unbiased screen.